Structural and functional analysis of the β-barrel domain of BamA from Escherichia coli

In gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a beta-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved component. To elucidate the mechanism of BamA-mediated OMP biogenesis, we determined the crystal structure of the C-terminal transmembrane domain of BamA from Escherichia coli (EcBamA) at 2.6 angstrom resolution. The structure reveals 2 distinct features. First, a portion of the extracellular side of the beta barrel is composed of 5 markedly short beta strands, and the loops stemming from these beta strands form a potential surface cavity, filled by a portion of the L6 loop that includes the conserved VRGF/Y motif found in the Omp85 family. Second, the 4 extracellular loops L3, L4, L6, and L7 of EcBamA form a dome over the barrel, stabilized by a salt-bridge interaction network. Functional data show that hydrophilic-to-hydrophobic mutations of the potential hydrophilic surface cavity and a single Arg547Ala point mutation that may destabilize the dome severely affect the function of EcBamA. Our structure of the EcBamA beta barrel and structure-based mutagenesis studies suggest that the transmembrane beta strands of OMP substrates may integrate into the outer membrane at the interface of the first and last beta strands of the EcBamA barrel, whereas the soluble loops or domains may be transported out of the cell via the hydrophilic surface cavity on dislocation of the VRGF/Y motif of L6. In addition, the dome over the barrel may play an important role in maintaining the efficiency of OMP biogenesis.